efficacy of vaccination on staphylococcus aureus and coagulase negative staphylococci intramammary infection dynamics in 2 dairy herds

J Dairy Sci 97:115 http://dx.doi.org/10.3168/jds.2014-8008 â american Dairy Science associationđ, 2014 Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in dairy herds Y H Schukken,*†1 V Bronzo,‡ C Locatelli,‡ C Pollera,§ N Rota,‡ A Casula,‡ F Testa,‡ L Scaccabarozzi,‡ Ricard March,# Daniel Zalduendo,# Roger Guix,# and P Moroni*‡ *Department of Population Medicine and Diagnostic Sciences, college of Veterinary Medicine, cornell university, Ithaca, nY 14853 †GD animal health, arnsbergstraat 7, 7418 eZ Deventer, the netherlands ‡università degli Studi di Milano, Dipartimento di Scienze Veterinarie per la Salute, la Produzione animale e la Sicurezza alimentare, via celoria 10, 20133 Milan, Italy §università degli Studi di Milano, Dipartimento di Scienze Veterinarie e Sanità Pubblica, via celoria 10, 20133 Milan, Italy #hipra S a laboratorios, avenida la Selva 135, amer (Girona), Spain ABSTRACT by visible changes in milk, including the presence of clots, flakes, serum, or even blood inclusion Subclinical mastitis is characterized by increased SCC, reduced milk production, and, in many cases, a higher risk of early removal from the farm Several preventative strategies have been applied to minimize the incidence of bovine mastitis, including optimization of milking procedures and milking hygiene, antibiotic therapies, vaccinations, segregation, and culling of persistently infected cows However, mastitis remains an important disease on many dairy farms and, due to the high costs of clinical mastitis, reduction in the severity of the symptoms of mastitis and obtaining a more rapid clearance of established infections is of great value to dairy farmers (Cha et al., 2011; Hertl et al., 2011) The severity of clinical symptoms of coliform mastitis has been shown to be reduced by immunization with commercially available J-5 bacterin (Wilson et al., 2007) The efficacy of this vaccine for the prevention of mastitis caused by Escherichia coli has been investigated in experimental challenge studies (Wilson et al., 2007) These studies implied that immunization with J-5 bacterin reduced the severity of local and systemic signs of clinical mastitis following intramammary challenge Efficacy of vaccination against Staphylococcus aureus and CNS is a very different concept than efficacy of vaccination against E coli (Torvaldsen and McIntyre, 2002) Whereas with E coli the vaccine is mostly expected to reduce severity of infection, with Staph aureus and CNS the vaccine is particularly valuable when vaccination results in a reduction of incidence and duration of infection, the key contributors to within herd infection dynamics (Schukken et al., 2011) Vaccines against staphylococci have been studied and suggested as an important tool in the management of staphylococcal infections in dairy cows (Pereira et al., 2011; Daum and Spellberg, 2012) Experimental challenge studies with Staph aureus have shown an effect of vaccination on the amount of bacterial shedding after The aim of this study was to evaluate vaccine efficacy of a commercial vaccine (Startvac, Hipra Spain) aimed at reducing intramammary infections (IMI) with Staphylococcus aureus and coagulase-negative staphylococci under field conditions During the 21-mo duration of the study, 1,156 lactations from 809 cows were enrolled in herds During the first phase of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (at ~6 mo) At that point, when 50% vaccination coverage was reached, cows that were due to calve were randomly assigned to be vaccinated or left as negative controls Cure rate, rate of new infection, prevalence, and duration of infections were analyzed Vaccination resulted in a moderate reduction in incidence of new staphylococcal IMI and a more pronounced reduction in duration of IMI associated with reduction of the basic reproduction ratio of Staph aureus by approximately 45% and of coagulase-negative staphylococci by approximately 35% The utilization of vaccine in combination with other infection-control procedures, such as excellent milking procedures, treatment, segregation, and culling of known infected cattle, will result in an important reduction in incidence and duration of intramammary staphylococcal infections Key words: Staphylococcus aureus, coagulase-negative staphylococci, intramammary infection, vaccine INTRODUCTION Mastitis is one of the most frequently occurring and costly diseases in dairy cows (Barkema et al., 2006; Halasa et al., 2007) Clinical mastitis is characterized Received February 2, 2014 Accepted April 15, 2014 Corresponding author: y.schukken@gddeventer.com Schukken et al challenge (Pérez et al., 2009); however, such experimental studies were not able to demonstrate a reduction in infection transmission Several study designs to estimate vaccine efficacy of contagious infections have been proposed (Haber et al., 1991; Halloran et al., 1991, 1997, 1998) Randomization can take place at either the herd or at the individual animal level To estimate the overall population vaccine efficacy using herd-level randomization, large numbers of vaccinated and control herds would be necessary Within-herd randomization of cows to vaccination and control has obvious study size benefits, but is hampered by the potential herd immunity provided by vaccinates to the control animals in the same herd (Halloran et al., 1991) Comingling of vaccinated and control cows allows the calculation of direct vaccine efficacy, but this estimate of vaccine efficacy will be biased toward zero This direct vaccine efficacy is an underestimation of the overall population vaccine efficacy due to the herd immunity of the vaccinated individuals that protect the unvaccinated controls (Halloran et al., 1991) However, instead of basing vaccine efficacy on infection incidence, vaccine efficacy can be estimated based on infection transmission and infection duration parameters (Halloran et al., 1997) These infection dynamics parameters can be estimated from precisely documented infections in comingled populations, and the resulting vaccine efficacy turns out to be an unbiased estimate of overall population vaccine efficacy as long as the analysis is controlled for total exposure experience (Lu et al., 2009) The number of vaccines against staphylococcal pathogens available on the market is small, and the efficacy of the results of these in peer-reviewed studies from commercial dairy farms is generally limited (Middleton et al., 2009) Recently, a combined staphylococcal and J5 E coli vaccine (Startvac, Hipra Spain), was introduced in the European market and, subsequently, in many other countries worldwide The staphylococcal component of the vaccine is based on a bacterin of Staph aureus strains with particular high cell wall components, such as exopolysaccharides, that may be involved in the biofilm phenotype of the bacteria (Harro et al., 2010; Prenafeta et al., 2010) To evaluate vaccine efficacy in the case of Staph aureus and CNS infections, the infection status of quarters of cows needs to be determined precisely over time (Halloran et al., 1997) Such precise data will allow the evaluation of vaccination on new IMI and IMI duration; at this point, few, if any, such studies have been reported in the literature The objective of the current trial was to evaluate vaccine efficacy under field conditions in herds with a known infection prevalence of Staph aureus and CNS Journal of Dairy Science Vol 97 No 8, 2014 MATERIALS AND METHODS Herds To evaluate vaccine efficacy we studied infection dynamics in herds with a total of approximately 450 dairy cows milking at any point in time The herds had a known prevalence of Staph aureus of at least 5% of cows and a bulk milk SCC between 250,000 and 400,000 SCC/mL Both herds used dry cow therapy on all quarters of all cows Clinical mastitis cases were treated according to herd-specific protocols that were similar but not identical Herd A had dedicated milkers that used a milking protocol with forestripping and wiping with single-use cloth towels Herd B had one dedicated milker that used forestripping and wiping with single-use paper towels Both herds used postmilking teat disinfection Culling decisions were made by the farm owners based on fertility and lameness criteria in both herds The trial started in May 2011, with sampling, vaccinating, and collection data gathered on the farms until February 2013 for farm A, for a total of 21 mo, and October 2012 for farm B, for a total of 18 mo Farm A maintained an average of 130 Holstein milking cows housed in freestall barns in deep-bedded cubicles with straw Farm B maintained an average of 320 Holstein milking cows housed in freestall barns in deep-bedded cubicles with sawdust On both farms, cows that were close to calving were moved to a loose-housing maternity pen bedded with straw Animals were housed for the first week of lactation in a large loose-housing pen with straw After wk of lactation, cows were moved to freestall facilities All groups of cows in both dairies were fed a balanced TMR in feed alleys with headlocks that allowed restraint of cows for examination and administration of treatments, medications, and vaccinations No segregation of cows based on IMI status or SCC level was done on either farm Milking Equipment Evaluation On Farm A, cows were milked in a double-12 parallel parlor times per day, whereas Farm B had a double-15 herringbone parlor and cows were also milked times per day On the farms, milking equipment was evaluated twice during the study period by technicians of the Regional Breeding Association using a complete ISO 6690:2007-defined evaluation (ISO, 2007) Equipment evaluation took place at the beginning and at approximately yr into the study No important concerns with milking equipment were identified on either farm 3 OUR INDUSTRY TODAY Cow Data Cow data on calving, parity, reproduction (AI dates, pregnancy), clinical disease (including retained placenta, endometritis, metritis, lameness, clinical mastitis, and metabolic diseases such as ketosis, abortion, and displaced abomasum), and culling was collected for all cows in the herd During the trial, Italian DHIA testing in both herds was done monthly for milk production, fat, protein, and SCC, but these data were not further analyzed for this report All breedings on both farms were done using AI Cow data were collected using a computerized herd record-keeping system (Dairy Comp 305, Valley Agricultural Software, Tulare, CA) Vaccination Vaccination took place according to label directions in the dry period and early lactation The first vaccination was at 45 d (±3 d) before the expected parturition date, the second vaccination at 35 d thereafter (±3 d), corresponding to 10 d before the expected parturition date, and the third vaccination was at 52 DIM (±3 d) No placebo or sham vaccination was used in this trial Cows going through a second dry period during the study were kept in the same treatment group (vaccinated or control) At the start of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (~6 mo) At that point in time, when 50% vaccination coverage was reached, cows were randomly assigned to be vaccinated or left as controls Trained farm personnel on farm A and the herd veterinarian on farm B performed all vaccinations Assignment of vaccination was done using the European cow registration number, whereby even-numbered cows were vaccinated and oddnumbered cows were kept as controls Cows were identified in each farm using unique farm-specific ear tags No logical relationship existed between the on-farm ear tag number and the official 13-digit European cow registration number We thereby assume that this was essentially a randomized controlled and single-blinded trial, as the herd staff was not aware of the vaccination status of the animals Milk Sampling Monthly quarter sampling of all lactating cows in herds was done during the trial period In addition, quarters were sampled by the farm staff when a case of clinical mastitis occurred, when cows were dried off, upon calving, and at culling Samplings related to dry off, calving, and culling were done within 24 h of the event Sampling in cases of clinical mastitis was done upon detection, before treatment was applied Before sampling, teat ends were carefully cleaned and disinfected with chlorhexidine First streams of foremilk were discharged, and then approximately 10 mL of milk was collected aseptically from each teat into sterile vials Samples were stored at 4°C until bacteriological assays and SCC tests were initiated immediately after arrival back in the laboratory Bacteriological Analysis Bacteriological cultures were performed according to standards of the National Mastitis Council (NMC, 1999) Ten microliters of each milk sample were spread on blood agar plates (5% defibrinated sheep blood) Plates were incubated aerobically at 37°C and examined after 24 h Colonies were provisionally identified on the basis of morphology, hemolysis patterns, and Gram staining Gram-positive organisms were differentiated in staphylococci and streptococci by the catalase reaction The coagulase tube test in rabbit plasma was used to differentiate Staph aureus and CNS species Catalase- and coagulase-positive bacteria were reported as Staph aureus, whereas catalase-positive and coagulase-negative species were reported as CNS Catalase-negative organisms had their identity confirmed by the API20Strep system (bioMerieux, Marcy l’Etoile, France), designed for Streptococcus spp identification Pathogens reported as other Streptococcus spp corresponded to species of streptococci that are less commonly reported in the literature or to pathogens that are not included in the API system identification panel Gram-negative bacteria were identified by oxidase test, as well as by growth characteristics onto MacConkey agar (Oxoid Ltd., Basingstoke, UK) and Eosin Methylene Blue agar (Oxoid Ltd.; http://www.oxoid.com/UK/blue/prod_ detail/prod_detail.asp?pr=CM0069&org=66) Further identification was performed with the API20E and API20NE system (bioMerieux, Marcy l’Etoile, France) Gram-negative bacteria with very low prevalence that could not be identified by the methods described were reported as “other gram-negative.” The numbers of each colony type were recorded Representative colonies were then subcultured on blood agar plates and incubated again at 37°C for 24 h to obtain pure cultures For plates with Staph aureus and CNS growth, the number of colonies was recorded for each species isolated, and colonies were reisolated and frozen for future characterization at −80°C in Nutrient Broth (Merck KGaA, Darmstadt, Germany) with 15% glycerol Journal of Dairy Science Vol 97 No 8, 2014 Schukken et al Definition of Infection Status Staphylococcus aureus was considered to cause an IMI if at least colony (≥100 cfu/mL) was isolated For CNS, IMI was defined by the isolation of at least colonies (≥200 cfu/mL) from a single sample or ≥100 cfu/mL from a clinical sample When multiple (at least out of 3) consecutive samples with ≥100 cfu/mL of CNS were identified, this was also considered an IMI These definitions are based on the consensus opinion of mastitis research workers as published by Dohoo et al (2011) and Andersen et al (2010) A quarter was defined as uninfected and at risk for a new infection when consecutive samples were culture-negative An infection was considered cured if consecutive monthly milk samples did not show the presence of the causative organism Milk samples where or more species were identified were considered contaminated All culture results were kept from both farm staff and herd veterinarians until the very end of the study When entering or leaving the trial, or reentering after calving, a single negative sample was considered sufficient to be defined as uninfected Statistical Analysis Data were analyzed using the SAS version 9.2 system (SAS Institute Inc., Cary, NC) Descriptive analysis was done on all important outcome variables and covariates Transformations were used where outcome variables were not normally distributed (e.g., SCC and cfu) Logistic Regression Analysis—Risk Factors for New IMI and Cure of IMI Linear regression models were used for analysis of crude prevalence and incidence of IMI In these generalized linear models the only data were used after the 50/50 randomization in the herds had started Every quarter-month at risk of either an incident or prevalent staphylococcal IMI contributed a line of data to this analysis The generalized linear model had the following format: Logit (Y) = intercept + MIM + lactgroup + herd + vaccination + complex error, where Y is the outcome of interest (incidence or prevalence of Staph aureus and CNS); MIM is months in milk; lactgroup is the lactation number of the cow, grouped into 1, 2, and 3+; herd is the herd code; and vaccination is either vaccinated or control Complex error is a correlated error term where within-cow correlation is combined with a random binomial error Journal of Dairy Science Vol 97 No 8, 2014 Relevant interactions were evaluated in the model and included when statistically significant Duration of infection was estimated with the use of time-to-event analysis Kaplan-Meier estimates of the survivor curves were used for graphical representation of the results Cox regression was used for estimating the effect of vaccination on the duration of infection For this analysis, only new infections were used that started after the 50/50 randomization in the herds had started Modeling Infection Dynamics The rate of new infections per day at risk was calculated for vaccinated and control cows The rates were calculated on a monthly basis (calendar months) for the duration of the trial For evaluation of vaccine efficacy of Staph aureus and CNS, the transmission rate (β), taking exposure into account, was calculated and compared between vaccinated and control cows Exposure was based on the number of Staph aureus- or CNS-shedding quarters at the same time in the herd No distinction was made between infected quarters in the same cow and the susceptible quarters and infected quarters in other cows The modeled relationship was defined as New Staph aureus or CNS infections(v/c) = β(v/c) × S(v/c) × (Iv + Ic) + covariates, where v/c is vaccinated or control; S is the number of susceptible quarters; I is the number of infected quarters; and β is the transmission parameter Vaccine efficacy for new infections may then be estimated as 1− (βv/βc) Similarly, cure of infection was modeled using Cure Staph aureus or CNS infections(v/c) = α(v/c) × I(v/c) + covariates, where α is the cure rate of infections Again, vaccine efficacy may then be estimated as − (αv/αc) Estimates of α and β were obtained through linear models using Poisson regression (see also Lam et al., 1996; Barlow et al., 2013) The regression model for estimation of β was ln (no of new infections v/c ) = b*v/c + covariates + offset, where the offset is given by ln {[Sv/c × (Iv + Ic)]/N}, where N is the total population size The parameter β can then be calculated as exp(β*) For estimation of α, the Poisson regression equation was OUR INDUSTRY TODAY ln (no of cured infections v/c ) = a*v/c + covariates + offset, where the offset is given by Ln (Iv/c) The parameter α was then calculated as exp(α*) The unit of analysis in both the regression analysis to estimate β*v/c and α*v/c was a calendar month All data were used in this analysis, and a covariate that measured the month to or since the 50/50 vaccination point was included as a covariate in the model Population vaccine efficacy was estimated using the parameters α and β, where vaccine efficacy were respectively defined as Vaccine efficacy for new infections = − βvaccinated , βcontrol whereas Vaccine efficacy for cure of infections = − α control α vaccinated Combining the information of parameters α and β into an overall infection reproduction ratio provides an unbiased summary parameter on vaccine efficacy The basic reproduction ratio (R0) was defined as R0 = β/α, and the resulting vaccine efficacy is then calculated as 1− R0,vaccinated R0,control = 1− (β / α )vaccinated (β / α )control The variance of R0 may be calculated from the sum of the variance of the logarithm of the components of R0: Var [ln (R0)] = Var (β*) + Var (α*) This overall efficacy parameter is expected to provide the best summary of the overall effect of vaccination on infection dynamics in a vaccinated population (Halloran et al., 2008) Samples Size The study was planned using a design of comingling vaccinates and controls with control per vaccinate As cow is the unit of vaccination, sample size calculations were performed at cow level Prior data indicated that the new infection rate among controls is approximately 0.15 per lactation This new infection risk of 0.15 includes both Staph aureus and CNS infections If the true vaccine efficacy is at least 50%, then the new infection rate for vaccinated cows is 0.075 (Dohoo, 2004) We needed to study at least 250 vaccinated cows and 250 control cows to be able to reject the null hy- pothesis that the new infection rates for vaccinated and control cows were equal (efficacy = 0) with probability (= power) 0.8 The Type I error probability associated with this test of this null hypothesis is 0.05 Because of the within-cow dependency due to comingling (Halloran et al., 1997), we estimated an increased sample size by approximately 25% resulting in at least 315 cows per treatment arm, resulting in a study size of at least 630 cows in total RESULTS Data Quality Data checks and data entry occurred throughout study Entry into the vaccination group was not as fast as expected on farm A, as pregnant heifers were initially not vaccinated This was corrected in the database as soon as it was noted For this reason, the farm reached the 50/50 point a few months later; thus, it was decided to keep the herd in the study for a longer period compared with farm B Data quality was checked throughout the study and additional information on incomplete data points was collected where needed Vaccination compliance was not always perfect during the trial; this is discussed in more detail herein Descriptive Statistics During the entire study, a total of 1,156 lactations in 809 cows were identified in both herds; 658 cows (56.92%) were enrolled as controls, 343 cows (29.67%) were fully vaccinated, and 155 cows (13.34%) started the vaccination but were not fully vaccinated due to calving date estimation errors in pregnancy checking, early pregnancy loss, abortions, early calving, or end of the study As vaccination was initially done on all cows calving into the lactating herd, the percentage of cows that were vaccinated increased rapidly in both herds The percentage of vaccinated lactations in each herd throughout the trial is shown in Figure In herd B, the 50/50 status was reached in mo of the study, whereas in herd A this was at 11 mo into the study Given that vaccinations start approximately mo before anticipated calving, the change in randomization procedure started in herd B at mo after the start of the study, whereas this was mo after the start of the study in herd A Bacterial Culture Results Throughout the study, 39,506 quarter milk samples were taken and used for bacterial culture The results of bacterial culture of all these samples are shown in Journal of Dairy Science Vol 97 No 8, 2014 Schukken et al Figure Percentage of lactations that were either vaccinated or control In herd A the 50/50 status was reached in mo 11 into the study, whereas in herd B this was at mo Table The most commonly isolated pathogens in herd A were Staph aureus (2,151; 15.6%) and CNS (937; 6.8%) In contrast, in herd B, CNS (1,139; 4.6%) were more frequently identified then Staph aureus (929; 3.8%) Culture-negative status was observed in 9,503 samples (69%) for farm A and in 19,936 samples (80.5%) for farm B Prevalence of Staph aureus during the course of the study remained more or less stable in farm A, ranging from 19.6% at mo to 14.8% at mo 22, Table Bacterial results of all samples collected during the trial, monthly samples, dry off, calving, culling, and clinical mastitis cases Farm A Pathogen Staphylococcus aureus CNS Streptococcus bovis Streptococcus canis Streptococcus dysgalactiae Streptococcus mitis Streptococcus uberis Streptococcus spp Corynebacterium spp Enterococcus faecalis Lactococcus lactis Aerococcus viridans Escherichia coli Enterobacter spp Other gram-negative pathogen Klebsiella spp Pasteurella spp Proteus spp Prototheca spp Serratia spp Trueperella pyogenes Bacilli Blind quarter Missing or contaminated Culture negative Total Journal of Dairy Science Vol 97 No 8, 2014 Farm B No Percent No Percent 2,151 937 19 14 132 89 40 38 11 58 81 19 52 63 260 671 9,503 14,159 15.6 6.8 0.0 0.0 0.1 0.1 1.0 0.6 0.3 0.3 0.1 0.4 0.6 0.1 0.4 0.0 0.0 0.5 0.0 0.0 0.0 0.0 1.9 2.0 69.0 100 929 1,139 50 176 36 217 117 63 55 70 88 191 17 36 116 65 15 26 539 1,452 19,936 25,347 3.8 4.6 0.2 0.0 0.7 0.1 0.9 0.5 0.3 0.2 0.3 0.4 0.8 0.1 0.1 0.5 0.0 0.3 0.0 0.1 0.0 0.1 2.1 3.5 80.5 100 OUR INDUSTRY TODAY Figure Prevalence of Staphylococcus aureus (top) and CNS (bottom) IMI in all quarters during the course of the study in vaccinated and control (dash-dotted line) cows Only cows that were eventually fully vaccinated were included in this analysis As per vaccination protocol, vaccinated cows received vaccinations at the start of lactation (2) and received the third and final dose (3) at approximately 53 DIM but reduced dramatically in farm B from 10.5% at mo to 1.2% at mo 18 In both farms, a fairly stable situation existed, without much change in prevalence of CNS IMI, ranging from 5.0% at mo to 9.2% at mo 22 for farm A and from 5.1% at mo to 4.4% at mo 18 for farm B When expressing prevalence by month in lactation, the data indicated a gradually increasing difference in prevalence between controls and vaccinates This trend was present and statistically significant for both Staph aureus and CNS IMI The least squares means of the prevalence of infection for Staph aureus and CNS is shown in Figures 2a and 2b Statistical Analysis Logistic Regression and Cox Regression Analysis—Risk Factors for New IMI and Cure of IMI Risk of new IMI with Staph aureus and CNS was analyzed by generalized linear regression analysis analyzing only new infections that occurred in cows calving after the 50/50 randomization had started The Journal of Dairy Science Vol 97 No 8, 2014 Schukken et al Table Final logistic regression models of new Staphylococcus aureus and CNS IMI1 Staph aureus Effect Intercept Staph aureus or CNS history Month in lactation               Lactation     Vaccination   Vaccination × month in lactation               Herd       Vaccinated Control B A CNS Estimate (SE) Pr > |t| Estimate (SE) Pr > |t| −3.48 (1.45) 0.60 (0.32) −1.24 (0.38) −0.90 (0.36) −0.68 (0.35) −0.29 (0.34) Baseline 0.15 (0.36) 0.19 (0.40) 0.32 (0.35) −1.62 (0.27) −0.19 (0.21) Baseline 0.14 (0.20) Baseline NS NS NS NS NS NS NS NS −2.84 (0.28) Baseline 0.02 0.06 0.00 0.01 0.05 0.40   0.68 0.64 0.36